The present invention relates to a method and composition for detecting a luminescent transition metal complex. In particular, the present invention is concerned with the detection of an analyte in an aqueous solution at a physiological pH using an electrochemical luminescent transition metal complex label.
There is a continuing and extensive need to detect and quantify various analytes in a test sample of a physiological fluid. An analyte can be a naturally occurring substance, such as an antibody, antigen, nucleic acid, enzyme, hormone or a metabolite or derivative thereof. An analyte can also be a manmade substance, such as a drug (including both therapeutic drugs and drugs of abuse) or a toxin or a metabolite or derivative thereof. A physiological fluid can be, for example, blood, serum, plasma, urine, amniotic, pleural or cerebrospinal fluid.
An analyte can be detected by labelling the analyte, an analyte analog or a binding partner for the analyte with a detectable label. Preferably, a label is easy to use, can be attached to a variety of substances, thereby enabling its use to detect a variety of analytes, is inexpensive, and has a distinctive and easily detectable signal, permitting rapid differentiation of the label from other substances, including other labels, and thereby analyte detection.
Radioactive labels have been used extensively to detect various analytes. Unfortunately, radioactive labels are expensive, hazardous to use, require sophisticated equipment, have a limited sensitivity, as well as a generally short shelf life, and have stringent disposal requirements.
Non-radioactive labels include labels detectable by spectrophotometric, spin resonance, and luminescence techniques. The use of luminescent material as a label can be advantageous because of high label sensitivity and specificity. Luminescence is a nonthermal emission of electromagnetic radiation by a material upon some form of excitation. The process of luminescence typically involves absorption of energy, excitation and emission of energy, usually in the form of radiation in the visible portion of the spectrum. The type of luminescence can be defined by the excitation means. Thus, electroluminescence is luminescence whereby the excitation source is an electric field. Chemiluminescence is luminescence wherein the excitation source is a chemical reaction.
Electrochemical luminescence (ECL) can occur when an electric field induces a chemical reaction which chemical reaction results in the desired luminescence. In typical ECL methodologies, a reactive species is generated from a stable precursor (i.e. a suitable label) at the surface of an electrode.
A variety of substances can be stimulated to emit detectable electromagnetic radiation via an electrochemical reaction. Some of these substances also have the characteristics, such as solubility and reactivity which can make them suitable for the labelling and detection of different analytes. Such a substance can be called an "ECL label".
Biological molecules, including many analytes present in a sample of a physiological fluid, are typically soluble only in aqueous solution. Additionally, many nonaqueous solvents, including diverse organic and inorganic solvents, can denature or damage labile analytes present in physiological fluids. It is also known that many organic solvents are carcinogenic, volatile and difficult to handle and to dispose of.
Furthermore, the particular configuration and structure of many analytes present in a sample of a physiological fluid necessitates that the aqueous solution in which an analyte is dissolved have a physiological pH, that is a pH between about 6 and about 8. Frequently, the physiological pH of the aqueous solution is maintained at about pH 7 to prevent denaturation or fragmentation of analytes from a physiological fluid.
Thus, most analytes of physiological interest are preferably detected when they are present in an aqueous solution which does not contain any organic or inorganic cosolvents, and which aqueous solution has a physiological pH.
Unfortunately, many substances which exhibit a detectable electrochemical luminescence, and which therefore have potential utility as analyte labels, cannot be used, or cannot be used efficiently, unless present in an organic solvent or an organic cosolvent (such as ether, benzene or acetonitrile), and/or with one or more additional compounds such as a peroxydisulphate or a peroxysulphate. Thus, these substances are unsuitable as ECL labels for the detection of analytes present in physiological fluids.
Various organometallic compounds, including a number of transition metal-organic ligand complexes, have been examined as potential ECL labels for the detection and quantification of analytes present in physiological fluids. Thus, organometallic complexes of ruthenium, osmium, rhenium and/or rhodium can be particularly attractive due, for example, to thermal, chemical and photochemical stability, high emission intensity (which can increase the detection sensitivity) and long emission lifetimes (which can allow use of less expensive measurement instrumentation) of such complexes. It is known that application of an oxidative potential (oxidative ECL) or of a reductive potential (reductive ECL) to a transition metal complex can be used to generate an electrochemical luminescence by the transition metal complex. Specifically, it is known that luminescence of a ruthenium tris-2,2'bipyridine complex (which can be expressed as Ru(bpy).sub.3.sup.2+) can be generated using chemical, photochemical, and electrochemical excitation means. Bard, A. J. and Whiteside, G. M., WO 86/02734, which publication is incorporated herein in its entirety.
Unfortunately, there are significant drawbacks and deficiencies with the methodology used in the prior art to generate ECL of a transition metal complex. These problems exist whether oxidative or reductive ECL processes are used to cause the transition metal complex to luminesce, as set forth below.
Oxidative ECL involves the application of a sufficiently positive electric potential to a solution containing a substance capable of participating in an electrochemical luminescence reaction. Bard and Rubinstein, in J. Am. Chem. Soc., (1981), 103, 512-516 discuss an electrochemical oxidation of a ruthenium bipyridyl complex in the presence of oxalate and other organic acids in water. Bard et al., obtained an oxidative ECL by applying a positive or anodic potential greater than about +0.9 volts to an electrode, either continuously or intermittently.
The oxidative ECL method was used by Bard et.al. to detect about 10.sup.-6 M of oxalate (Anal. Chem., (1983), 55, 1580-1582) and about 10.sup.-9 M of the ruthenium ECL label (Anal. Chem., (1984), 56, 2413-2417, see also WO 86/02734). For Bard et al., no luminescence was reported when a cathodic or negative potential between about 0.0 volts and about -2.0 volts was applied to the reagent solution. Additionally, the oxidative ECL reaction was carried out at a nonphysiological acidic pH to obtain optimal luminescent intensity.
Oxidative ECL in an aqueous buffer solution at a physiological pH has been reported: Leland, J. K. and Powell, M. J., (J. Electrochem. Soc., (1990), 137(10), 3127-3133, and WO 90/05296, May 17, 1990) (which publications are incorporated herein in their entireties) discuss detection of an ECL reaction which utilizes the oxidation of an amine such as tripropylamine and a ruthenium bipyridyl complex as the luminophore. Unfortunately, light emission was observed by Leland et al., even in the absence of the ruthenium complex. Such a background or blank light emission in the oxidative ECL process limits analyte detection to about 100 pM.
Additionally, Noffsinger, J. B. and Danielson (Anal. Chem., (1987), 59, 865-868), and Brune, S. N. and Bobbit, D. R. (Anal. Chem., 1992, 64, 166-170) discuss an oxidative ECL method using various amines and a ruthenium bipyridyl complex. The excited state ruthenium was generated electrochemically, but the luminescence was obtained by subsequent mixing with an amine.
Oxidative ECL has also been discussed by Kenten, J. H., in "Rapid Electrochemiluminescence Assays of Polymerase Chain Reaction Products", Clin. Chem. 37/9, 1626-1632 (1991), where a tris (2, 2'-bipyridine) ruthenium (II) complex was used as a DNA probe label (available from IGEN, Inc., as the ORIGEN label), and in Kenten, J. H., et. al., in "Improved Electrochemiluminescence Label for DNA Probe Assays: Rapid Quantitative Assays of HIV-1 Polymerase Chain Reaction Products", Clin Chem. 38/6, 873-879 (1992), where, a transition metal ECL label comprising a ruthenium polypyridyl chelate (specifically, a tris ruthenium bipyridyl chelate) was used as a DNA probe by attaching the ruthenium complex to the oligonucleotide.
Thus, transition metal complexes have been used as detectable labels for various analytes in oxidative ECL processes. See also Blackburn, G. F., et. al., in "Electrochemiluminescence Detection for Development of Immunoassays and DNA Probe Assays for Clinical Diagnostics", "Clin. Chem. 37/9, 1534-1539 (1991), which publication is incorporated herein in its entirety.
Unfortunately, there are significant problems with the use of an oxidative ECL process to detect an analyte present in a sample of a physiological fluid. Generally, the positive voltage potential required to obtain oxidative ECL can result in the evolution of oxygen gas, with resulting deleterious effects to the electrodes used to apply the oxidative potential. Thus, the relatively high positive or anodic oxidative potential required to obtain oxidative ECL can result in the electrodes becoming pitted and eventually dissolved, due at least in part to the oxygen gas evolution.
Additionally, the oxygen gas typically evolved during an oxidative ECL process can remain at or near the electrode surface as a layer of small bubbles lining and insulating the electrode. Such a layer of trapped gas can significantly increase the electrochemical impedance or resistance between the electrodes and can thereby act to prevent or to significantly attenuate the inducement of an ECL reaction at a given electrical potential applied to the aqueous solution. The presence of trapped oxygen gas bubbles over the electrodes can also cause electric arcing between the working and counter electrodes, thereby making analyte detection difficult or impossible.
ECL which might be generated by electrochemical reduction (reductive ECL), less a nodic (oxidative) Potential and electro catalysis have therefore been examined as a way to overcome the problems of analyte detection present with known oxidative ECL processes. Reductive ECL is carried out by applying a sufficiently negative potential to the reagent solution. Such ECL can result in a higher signal output (i.e., more or a more intense light emission) per unit concentration of a stimulated metal chelate. This can allow a higher sensitivity and hence a lower analyte detection limit.
Significantly, the occurrence of oxygen gas evolution with resulting problems of accelerated electrode deterioration and solution impedance increase, is entirely absent or much less significant with reductive ECL, as compared to oxidative ECL processes.
White, H. S. and Bard, A. J., (J. Am. Chem. Soc., (1982), 104, 6891-6895) describe a reductive ECL in a 50% aqueous solution containing an organic co-solvent (such as acetonitrile), peroxydisulfate (a strong oxidizing agent) and a ruthenium bipyridyl complex as the luminophore.
Ege, D. et al., used the method of White et al., for the determination of about 10.sup.-13 M ruthenium (Anal. Chem. 1984, 56, 2413-2417, see also WO 86/02734). The reductive ECL reaction was carried out in a 50% acetonitrile/water mixture containing peroxydisulphate. Additionally, to observe any light emission and be able to detect a concentration below about 10.sup.-8 M, deaeration of the sample solution was required to remove residual oxygen.
Yamashita, K. P., et al., in "Direct Current Electrogenerated Chemiluminescence. Micro Determination of Peroxydisulfate in Aqueous Solution", Anal Chem. (1991), 63, 872-876, discusses the reductive ECL of a Ru(bipyrazine).sub.3 chelate in aqueous solutions containing 0.1M sodium sulfate. No ECL was observed in the absence of the peroxydisulfate. Significantly, Yamashita, et al., had to prepare solutions of Ru(bipyrazine).sub.3 and peroxydisulfate in the dark to avoid photochemical reaction between the reagents.
Furthermore, White, H. S. and Bard, A. J., (J. Am. Chem. Soc., (1982), 104, 6891-6895) describe a reductive ECL method based on pulsing a platinum electrode repetitively between -0.5 volts and -2.0 volts, using a Ruthenium chelate as the luminophore, in acetonitrile or a partially aqueous solution containing acetonitrile (1:1 by volume) and peroxydisulfate. ECL was not observed in aqueous solutions or in the absence of peroxydisulfate.
Thus, the current art of reductive ECL also has significant shortcomings. As set forth above, the requirements for, at least, one or more organic cosolvents or strong oxidizing agents such as peroxydisulphate in the reagent solution, and deaeration of the solution to allow analyte detection at even a modestly low concentration, severely limits the application of reductive ECL processes for the detection of analytes present in a sample of a physiological fluid below an analyte concentration of about 10.sup.-9 M.
Furthermore, although extensive work on reductive ECL processes has been carried out, there has been no report in the art, to the Applicants knowledge, of reductive ECL of a transition metal complex being accomplished in an aqueous solution at a physiological pH, permitting detection below about 10.sup.-9 M concentration without the addition of a strong oxidant such as peroxydisulfate and/or removal of dissolved oxygen from the aqueous solution.
It is known that the electrochemical luminescence can be enhanced by various compounds. For example, the electrochemical luminescence of phenyl acridinium-9-carboxylate (as a fluorosulfonate salt) can be enhanced by the addition of cetylammonium bromide to solutions of the acridinium ester. However, optimum electrochemiluminescence has required an elevated pH of from about pH 9 to about pH 12. Littig, J., S., et al., "Quantification of Acridinium Esters Using Electrogenerated Chemiluminescence and Flow Injection" Anal. Chem. (1992), 64, 1140-1144.
What is needed therefore is a method and composition for conducting reductive ECL of a transition metal label in an aqueous solution at a physiological pH. Preferably, the method and composition can permit detection of an analyte below an analyte concentration of about 10.sup.-9 M concentration. More preferably, the method can be accomplished without addition of a strong oxidant such as peroxydisulfate to, and/or removal of dissolved oxygen from, the aqueous solution. It would also be advantageous to be able to enhance both reductive and oxidative ECL by a transition metal label through the addition of one or more substances to the aqueous solution prior to stimulation of the transition metal label.